ABSTRACT The central focus of this competitive renewal application is to firmly examine a specific causal role of endothelial dihydrofolate reductase (DHFR) deficiency in abdominal and thoracic aortic aneurysm formation using new lines of genetic mice (>12, out of which 9 are produced in house), and to identify novel therapeutic strategies based on recent discoveries of mechanistic details of DHFR regulation. An innovative biomarker to monitor aneurysm growth and response to treatment will also be characterized. During the previous funding cycle we have been very productive in completing all of the proposed aims, demonstrating a novel causal role of endothelial nitric oxide synthase (eNOS) uncoupling in abdominal aortic aneurysm (AAA) formation. We have also extended our studies to examination of an essential regulatory role of endothelial DHFR gene in vascular homeostasis and aneurismal development employing several new mouse lines, development of novel strategies to restore eNOS function to prevent AAA by approaches of gene therapy and pharmacological interventions targeting endothelial DHFR, identification of a clinically relevant biomarker role of circulating tetrahydrobiopterin (H4B) in mice and humans to monitor aneurysms, as well as investigation of applicability to thoracic aortic aneurysm (TAA) of the novel molecular mechanisms identified for AAA, so that some newly established therapeutic options for AAA can be extended to management of TAA. These studies are considered highly significant and translational, as there has not been any oral medicine for aneurysm treatment, besides surgical correction of large aneurysms that is also associated with considerable risk. Novel strategies for early diagnosis, monitor and treatment of aneurysms would benefit a large population of patients that accounts for 9% of the population over than 65 year old. The following four specific aims will be addressed in depth for these important missions: Aim 1, to examine a specific and essential role of endothelial DHFR deficiency in aneurysm formation using global and endothelium-specific DHFR knockout mice, and endothelium-specific DHFR transgenic mice crossed with hph-1. Effects on AAA formation of novel DHFR upregulators will also be examined. Aim 2, to determine whether pathways attenuating Ang II/oxidative stress inhibition of DHFR are of preventive and therapeutic value in aneurysm formation. Pathways mediating H2O2 regulation of DHFR or BP lowering drugs will be targeted for these studies. Aim 3, to evaluate a biomarker role of circulating tetrahydrobiopterin in aneurysm formation and treatment using Ang II infused hph-1 and apoE null mice, and two patient populations. Aim 4, to examine whether uncoupling of eNOS also mediates TAA formation downstream of TGF? pathway, hence the newly identified AAA treatment strategies in Aims 1-3 are effective in treating TAAs as well. Accomplishments of these aims will uniquely provide novel mechanistic and therapeutic insights into development and management of aortic aneurysms.